BMSCs移植对缺血性脑卒中大鼠IL-1β、TNF-a表达的影响
发布时间：2018-08-22 17:00
【摘要】：目的 骨髓间充质干细胞（bone marrow mesenchymal stem ceIIs,BMSCs）移植治疗大脑中动脉阻塞(Middle CerebralArtery Occlusion，MCAO)模型鼠后，在不同时间点采用western blot、 ELISA方法检测IL-1β、TNF-α的表达变化，分析BMSCs移植对缺血性脑卒中后炎性因子的影响，探讨BMSCs移植治疗缺血性脑卒中修复受损神经功能的可能抗炎机制。 方法 取幼年雄性SD大鼠骨髓腔细胞，通过全骨髓培养法和细胞贴壁培养法体外分离、培养、扩增得到BMSCs，传至第4代，免疫荧光法鉴定，诱导分化为成骨细胞、脂肪细胞及神经干细胞并鉴定。取成年雄性SD大鼠，用Longa线栓法制造MCAO模型。参考Bederson JB评分法测评大鼠神经功能缺损，筛选入组MCAO模型鼠；TTC染色法显示脑组织梗死范围，剔除TTC（-）或有出血的大鼠样本。 取造模成功的MCAO模型鼠完全随机分为2组：A组/MCAO模型组（n=16），即建立MCAO模型后注射1ml生理盐水；B组/实验组（n=16），即建立MCAO模型后通过尾静脉注射法移植1ml2×l09/L BMSCs；A、B组各分为处理后1d、3d、7d、14d时间点亚组（n=4）。取正常大鼠设C组/空白组（n=4），不予任何干预。各亚组行心脏取血，ELISA方法检测大鼠血清IL-1β、TNF-α的表达变化；生理盐水灌注取脑，western blot方法检测大鼠脑组织IL-1β、TNF-α的表达变化。 结果 1.成功培养得到BMSCs。镜下细胞形态：原代BMSCs呈圆形或类圆形，贴壁数量少；经过纯化、扩增，贴壁细胞增多，呈长梭形，集落样生长，局部呈漩涡状；至P7代，细胞逐渐老化，以扁平细胞为主。细胞免疫荧光法鉴定：CD54（+）、CD34（-），符合BMSC免疫表型特点。BMSCs诱导为成骨细胞见细小颗粒于培养液；诱导为脂肪细胞见红色脂滴于胞浆；诱导为神经干细胞见棕色颗粒于胞浆。 2.大鼠神经功能评分：造模后1天实验组评分较模型组无明显差异（P0.05），3天、7天、14天实验组较模型组均有下降（P0.05），差异有统计学意义。 3.大鼠血清IL-1β、TNF-α表达（单位：ng/L）：1天、3天、7天、14天各亚组：IL-1β浓度为模型组27.0±0.35、29.2±0.60、25.2±0.65、22.8±0.66，，实验组25.2±0.31、26.8±0.56、20.8±0.39、19.2±0.94，空白组10.9±0.54；TNF-α浓度为模型组231±1.46、225±4.98、216±4.16、198±4.61，实验组189±2.85、178±3.17、166±5.27、157±6.03，空白组146±6.86；模型组较空白组均明显增多（P0.05），实验组较模型组均减少（P0.05），但较空白组仍增多（P0.05）。模型组及实验组与空白组比较：IL-1β表达1天增多，3天达高峰，7天下降，14天持续下降，但仍高于空白组（P0.05）；TNF-α表达1天增多达高峰，3天下降，7天、14天缓慢下降，但仍高于空白组（P0.05），差异有统计学意义。 4.大鼠脑组织IL-1β、TNF-α表达（灰度值）：1天、3天、7天、14天各亚组：IL-1β为模型组5502±108.9、6267±160.5、4425±158.1、3352±187.1，实验组4217±143.7、4706±111.8、3235±150.7、2307±120.7，空白组1874±70.7； TNF-α为模型组7801±148.9、6296±170.7、5399±240.9、3458±180.2；实验组6468±180.9、5789±119.7、4647±207.3、2828±152.6，空白组2215±159.5；模型组较空白组均增高（P0.05），实验组较模型组均降低（P0.05）较空白组增高（P0.05）。模型组及实验组与空白组比较：IL-1β表达1天增高，3天达高峰，7天下降，14天持续下降但仍高于空白组（P0.05）；TNF-α表达1天增高达高峰，3天下降，7天、14天缓慢下降，但仍高于空白组（P0.05），差异有统计学意义。 结论 1.缺血性脑卒中模型鼠中IL-1β、TNF-α表达增加，其神经损伤可能与炎症反应有关。 2.骨髓间充质干细胞移植可降低MCAO模型鼠IL-1β、TNF-α的表达，其修复神经功能的作用可能与BMSC下调炎性因子减轻炎症反应有关。
[Abstract]:objective
After transplantation of bone marrow mesenchymal stem cells (BMSCs) for treatment of middle cerebral artery occlusion (MCAO) model mice, the expression of IL-1 beta and TNF-a was detected by Western blot and ELISA at different time points. The effect of BMSCs transplantation on inflammatory factors after ischemic stroke was analyzed. Objective to investigate the possible anti inflammatory mechanism of BMSCs transplantation in repairing damaged neural function after ischemic stroke.
Method
Bone marrow cavity cells of young male SD rats were isolated, cultured and amplified in vitro by whole bone marrow culture and cell adherence culture. BMSCs were identified by immunofluorescence. The adult male SD rats were induced to differentiate into osteoblasts, adipocytes and neural stem cells and identified. MCAO model was established by Longa thread plug method. Neurological deficits were assessed by on-JB scoring method, and MCAO model rats were selected. The infarct size of cerebral tissue was shown by TTC staining, and TTC (-) or bleeding rat samples were excluded.
MCAO model rats were randomly divided into two groups: group A / MCAO model group (n = 16), 1 ml normal saline was injected after establishing MCAO model; group B / experimental group (n = 16), 1 ml 2 xl09 / L BMSCs were transplanted by tail vein injection after establishing MCAO model; group A and group B were divided into 1, 3, 7 and 14 day subgroups after treatment (n = 4). No intervention was given to the control group (n=4). Blood samples were taken from the heart of each subgroup. The expression of IL-1beta and TNF-alpha in serum was detected by ELISA, and the brain was perfused with normal saline, and the expression of IL-1beta and TNF-alpha in brain tissue was detected by Western blot.
Result
1. BMSCs were successfully cultured. The morphology of primary BMSCs was round or quasi-circular, with few adherent cells; after purification, amplification, adherent cells increased, spindle-shaped, colony-like growth, local vortex; to P7 generation, cells gradually aging, mainly flat cells. Identification of cell immunofluorescence: CD54 (+), CD34 (-), in line with BMSC immunity. BMSCs were induced into osteoblasts with fine granules in culture medium, adipocytes with red lipid droplets in cytoplasm, neural stem cells with brown granules in cytoplasm.
2. Rat neurological function score: There was no significant difference between the experimental group and the model group on the first day after modeling (P 0.05). The scores of the experimental group on the third day, the seventh day and the fourteenth day were lower than those of the model group (P 0.05).
3.The expression of IL-1betand TNF-alphain serum of rats (unit: ng/L):1 day, 3 days, 7 days, 7 days, 14 days:1 day, 3 days, 7 days, 14 days:the concentration of IL-1 Betin the model group was 27.0 [0.35, 29.2 [0.60, 25.2 [0.60, 25.2 [0.65, 22.2 [0.65, 22.8 [0.66, 22.8] 0.66, experimental group 25.2 [0.2 [0.31, 26.8 [0.56, 20.8 [0.39, 19.39, 19.2 [.2 [0.94.94] 94, blankgroup 10.9 198 + 4.61, experimental group Compared with the blank group, the expression of IL-1 beta in the model group and the experimental group increased in one day, reached a peak in three days, decreased in seven days and continued to decline in 14 days, but still higher than the blank group (P 0.05). In the blank group (P 0.05), the expression of TNF-alpha reached the peak in one day, decreased in three days, slowly decreased in seven days and 14 days, but still higher than the blank group (P 0.05), the difference was statistically significant.
4.Expression of IL-1 beta and TNF-alphain rat brain tissues (gray value):1 day, 3 days, 7 days, 7 days and 14 days:1 day, 7 days, 14 days:1 day, 3 days, 7 days, each subgroup:IL-1 betwas 5502 [108.9, 6267 [160.5, 4425 [160.5, 4425 [158.5 [158.1, 3352 [188.1, 3352 [187.1,3352].1.1,3352 [[4 17 [143.7.7.7,4706 [111.8, 4706 [111.8, 3235 [150.7, 2307 [150.7, 2307 [180.2; solid Compared with the blank group, the expression of IL-1 beta in the model group and the experimental group increased in one day, reached a peak in three days, decreased in seven days, but remained higher in 14 days. In blank group (P 0.05), the expression of TNF-alpha increased as high as 1 day, decreased on 3 days, decreased slowly on 7 days and 14 days, but still higher than blank group (P 0.05), the difference was statistically significant.
conclusion
1. The expression of IL-1beta and TNF-alpha increased in ischemic stroke model rats, and the nerve injury may be related to inflammation.
2. Bone marrow mesenchymal stem cell transplantation can reduce the expression of IL-1beta and TNF-alpha in MCAO model mice, and its effect on repairing nerve function may be related to the reduction of inflammatory factors by BMSC.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
本文编号：2197753
[Abstract]:objective
After transplantation of bone marrow mesenchymal stem cells (BMSCs) for treatment of middle cerebral artery occlusion (MCAO) model mice, the expression of IL-1 beta and TNF-a was detected by Western blot and ELISA at different time points. The effect of BMSCs transplantation on inflammatory factors after ischemic stroke was analyzed. Objective to investigate the possible anti inflammatory mechanism of BMSCs transplantation in repairing damaged neural function after ischemic stroke.
Method
Bone marrow cavity cells of young male SD rats were isolated, cultured and amplified in vitro by whole bone marrow culture and cell adherence culture. BMSCs were identified by immunofluorescence. The adult male SD rats were induced to differentiate into osteoblasts, adipocytes and neural stem cells and identified. MCAO model was established by Longa thread plug method. Neurological deficits were assessed by on-JB scoring method, and MCAO model rats were selected. The infarct size of cerebral tissue was shown by TTC staining, and TTC (-) or bleeding rat samples were excluded.
MCAO model rats were randomly divided into two groups: group A / MCAO model group (n = 16), 1 ml normal saline was injected after establishing MCAO model; group B / experimental group (n = 16), 1 ml 2 xl09 / L BMSCs were transplanted by tail vein injection after establishing MCAO model; group A and group B were divided into 1, 3, 7 and 14 day subgroups after treatment (n = 4). No intervention was given to the control group (n=4). Blood samples were taken from the heart of each subgroup. The expression of IL-1beta and TNF-alpha in serum was detected by ELISA, and the brain was perfused with normal saline, and the expression of IL-1beta and TNF-alpha in brain tissue was detected by Western blot.
Result
1. BMSCs were successfully cultured. The morphology of primary BMSCs was round or quasi-circular, with few adherent cells; after purification, amplification, adherent cells increased, spindle-shaped, colony-like growth, local vortex; to P7 generation, cells gradually aging, mainly flat cells. Identification of cell immunofluorescence: CD54 (+), CD34 (-), in line with BMSC immunity. BMSCs were induced into osteoblasts with fine granules in culture medium, adipocytes with red lipid droplets in cytoplasm, neural stem cells with brown granules in cytoplasm.
2. Rat neurological function score: There was no significant difference between the experimental group and the model group on the first day after modeling (P 0.05). The scores of the experimental group on the third day, the seventh day and the fourteenth day were lower than those of the model group (P 0.05).
3.The expression of IL-1betand TNF-alphain serum of rats (unit: ng/L):1 day, 3 days, 7 days, 7 days, 14 days:1 day, 3 days, 7 days, 14 days:the concentration of IL-1 Betin the model group was 27.0 [0.35, 29.2 [0.60, 25.2 [0.60, 25.2 [0.65, 22.2 [0.65, 22.8 [0.66, 22.8] 0.66, experimental group 25.2 [0.2 [0.31, 26.8 [0.56, 20.8 [0.39, 19.39, 19.2 [.2 [0.94.94] 94, blankgroup 10.9 198 + 4.61, experimental group Compared with the blank group, the expression of IL-1 beta in the model group and the experimental group increased in one day, reached a peak in three days, decreased in seven days and continued to decline in 14 days, but still higher than the blank group (P 0.05). In the blank group (P 0.05), the expression of TNF-alpha reached the peak in one day, decreased in three days, slowly decreased in seven days and 14 days, but still higher than the blank group (P 0.05), the difference was statistically significant.
4.Expression of IL-1 beta and TNF-alphain rat brain tissues (gray value):1 day, 3 days, 7 days, 7 days and 14 days:1 day, 7 days, 14 days:1 day, 3 days, 7 days, each subgroup:IL-1 betwas 5502 [108.9, 6267 [160.5, 4425 [160.5, 4425 [158.5 [158.1, 3352 [188.1, 3352 [187.1,3352].1.1,3352 [[4 17 [143.7.7.7,4706 [111.8, 4706 [111.8, 3235 [150.7, 2307 [150.7, 2307 [180.2; solid Compared with the blank group, the expression of IL-1 beta in the model group and the experimental group increased in one day, reached a peak in three days, decreased in seven days, but remained higher in 14 days. In blank group (P 0.05), the expression of TNF-alpha increased as high as 1 day, decreased on 3 days, decreased slowly on 7 days and 14 days, but still higher than blank group (P 0.05), the difference was statistically significant.
conclusion
1. The expression of IL-1beta and TNF-alpha increased in ischemic stroke model rats, and the nerve injury may be related to inflammation.
2. Bone marrow mesenchymal stem cell transplantation can reduce the expression of IL-1beta and TNF-alpha in MCAO model mice, and its effect on repairing nerve function may be related to the reduction of inflammatory factors by BMSC.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
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